Multicolor flow cytometry analysis of peripheral blood mononuclear cells from patients with sarcoidosis associated uveitis reveals novel cytokine and lymphocyte subset disease contributors

Abstract

Purpose : The precise pathogenesis of sarcoidosis and associated uveitis is still under investigation. The potential contribution of mucosal-associated lymphoid subsets including mucosal-associated invariant T (MAIT) cells and innate lymphoid cells (ILCs) is relatively unexplored. We developed a novel multi-parametric flow cytometry panel in this study to identify immune dysregulation of these signatures in patients with uveitis secondary to sarcoidosis.

Methods : Antibody panels examined cytokine production (granulocyte-macrophage colony-stimulating factor (GM-CSF); IL-17A; IL-17F; IL-22 and interferon-γ (IFNγ)) by MAIT cells, ILCs, and conventional CD4/CD8 T cells. Peripheral blood mononuclear cells (PBMC) from sarcoidosis patients with a concomitant active or inactive history of uveitis (n=16), and healthy controls (n=15) were polyclonally stimulated with phorbol myristate/ionomycin for 4 hours. Cells were then stained for flow cytometry and data acquired by FACS Symphony were analyzed in FlowJo (Tree Star Inc.) and Prism 8 (GraphPad). MAIT cells were identified either by having positive markers of TCR Va7.2 and CD161 or by MR1-5OP-RU tetramers. Outliers were removed and the Mann-Whitney U test was used for comparisons of the two groups.

Results : MAIT cells exhibited markedly increased GM-CSF (p=0.012) and IFNγ production (p=0.041) in sarcoid uveitis patients. For conventional lymphocytes, we observed a significantly increased frequency of IL-17F+ and interferon γ+ CD8+ T cells in sarcoid uveitis patients. We also observed a significantly increased frequency of GM-CSF+ CD4 T cells in sarcoid uveitis patients vs healthy controls. No significant differences were noted in the ILCs.

Conclusions : Our data suggest that IFNγ and RORγt-dependent cytokines, GM-CSF and IL-17F, are dysregulated in immune cell subsets in sarcoid uveitis. To our knowledge, this is the first study to implicate MAIT cells in sarcoid uveitis pathogenesis and is consistent with a gut-eye or lung-eye association contributing to uveitis.

This is a 2020 ARVO Annual Meeting abstract.

 

Figure 1. Frequency of cytokine production by stimulated MAIT cells which express the TCR V alpha 7.2 and CD161 or identified by MR1-5OP-RU tetramer. HC = healthy controls. *p < .05.